In vivo semen-associated pH neutralization of cervicovaginal secretions.

Physiological cervicovaginal acidity can partly inactivate human immunodeficiency virus (HIV). Basic semen components should be able to partially neutralize in vivo cervicovaginal pH. The goals of the study were to evaluate the relationship between cervicovaginal pH and presence of semen components in sexually active African women and to assess whether vaginal douching with water performed just after sexual intercourse could significantly reduce semen components and restore physiological cervicovaginal pH. Cervicovaginal secretion (CVS) from 56 heterosexual African women (19 to 45 years old), living in Bangui, Central African Republic, were evaluated for pH, semen components (prostatic acid phosphatase [PAP] and prostatic specific antigen [PSA]), cellularity, and hemoglobin at inclusion and after vaginal douching with 100 ml of water by using a bock. Before douching, semen components were found in 46 of 56 CVS (82%). The mean vaginal pH was 5.2 (range, 3.6 to 7.7), and concentrations of both PAP and PSA correlated positively and strongly with cervicovaginal pH (P < 0.001). After douching, semen components were found in 35 of 56 CVS (62%) (P = 0.03). Cervicovaginal PAP and PSA levels were significantly decreased (respectively, P < 0.0001 and P < 0.01; PAP, -72%; PSA, -87%), as was the total cell count (-60%; P < 0.0001). Furthermore, in CVS previously positive for both PAP and PSA, the mean vaginal pH was significantly decreased (6.5 versus 5.3, P < 0.01); no genital bleeding was observed. Frequent persistence of semen in CVS from heterosexually active African women leads to a shift from acidity to neutrality that could favor male to female HIV transmission. Vaginal douching provides significant elimination of semen after sexual intercourse; it should be considered for study as a supplementary means for the prevention of heterosexual HIV transmission.     

Inhibition of the lytic activity of perforin (cytolysin) and of late complement components by proteoglycans

The complement components (C6, C7, C8 and C9) implicated in the lysis of target cells and the pore-forming, lytic protein from cytotoxic T-lymphocytes and NK-cells, perforin, contain an amino acid sequence which is highly homologous to a repeat unit identified in the LDL-receptor (Tschopp et al., 1986, Nature, 322, 831-834). The domain of the LDL-receptor, which is thought to interact with a positively charged segment of its ligands apoprotein B and E, is rich in cysteine residues and contains a cluster of negative charges. We show that the negatively charged molecules suramin and glycosaminoglycans, the positively charged peptides protamine and polylysine, all of which are known to abolish binding of LDL to its receptor (Goldstein et al., 1985, A. Rev. cell. Biol., 1, 1-39) inhibit the lytic activities of C6, C7, C8, C9 and perforin. Moreover, these negatively charged molecules are potent inhibitors of cytolytic T-lymphocyte-mediated lysis of target cells, suggesting a functionally crucial role for perforin in cell-mediated cytolysis. We propose that the negatively charged, cysteine-rich domain of these complement proteins and perforin interacts with an as yet unidentified positively charged segment of its ligand in a manner analogous to the LDL-LDL receptor interaction. Homologous cysteine-rich domains in functionally unrelated proteins may therefore be functionally conserved as ideal rigid interaction domains with the conserved cysteine residues as framework. Specificity of the domain for its ligand would be conferred by the non-conserved amino acid residues.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3-) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3- LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.     

Inactivation of African horse-sickness virus by betapropiolactone and by pH

The inactivation of several types of African horse sickness virus (AHSV) by pH and by betapropiolactone (BPL) was studied. At 19 degrees - 22 degrees C the virus was stable between pH 6.0 and 10.4, whether suspended in mouse brain or in serumfree buffer. Below pH 5.6 and above pH 10.9, more than 99 per cent of infectivity was inactivated within 15 minutes. The addition of 50 per cent serum did not influence pH stability. Disinfection in the presence of citric acid and caustic soda is briefly discussed. Inactivation by BPL was complete within 30 minutes at 37 degrees C, yet incomplete after 15 hours at 4 degrees C. Types 3 and 9 virus grown in suckling mouse brain and types 1, 3 and 9 produced in pig kidney cells were equally susceptible to 0.1 per cent BPL, more than 99.9 per cent being inactivated. The effectiveness of BPL was reduced at least 10-fold by the addition of 50 per cent serum. No infective virus was detected following incubation of either tissue culture virus with 0.2 per cent BPL or of mouse brain virus with 0.3 per cent BPL. Virus suspensions exposed to 0.3 per cent BPL required buffering with Tris of at least 0.05 molar strength in order to maintain the pH within an acceptable range. Inactivated antigens prepared with 0.4 per cent or lower concentrations of BPL were immunogenic in guinea pigs.     

Calcium is essential for both the membrane binding and lytic activity of pore-forming protein (perforin) from cytotoxic T-lymphocyte

The influence of Ca on the membrane binding and lytic activity of lymphocyte pore-forming protein (perforin) was studied. In the absence of Ca, perforin did not bind to the target membranes and did not support lysis of the target cells. In contrast, in the presence of Ca perforin was able to bind to the cell membrane (Km greater than 0.2 mM). Almost all the perforin molecules bind to the membrane within 1 min at 0 degrees C. The addition of EDTA abolished the binding, indicating that the effects of Ca on the membrane binding are reversible. On the other hand, the perforin-mediated lysis of target cells was temp-dependent and also required the presence of Ca in the reaction mixture (Km = 0.05 mM). The difference between the Km values for the membrane binding and lytic activity suggests the presence of two distinct Ca-requiring steps in perforin-mediated target cell lysis.     

Extracellular acidic pH modulates oxygen-dependent cytotoxic responses mediated by polymorphonuclear leucocytes and monocytes.

In the present study, we compared the ability of human neutrophils and monocytes to display oxygen-dependent cytotoxic responses at pH 7.4 and 6.2. Our results show that cytotoxicity induced by immune complexes (IC), zymosan, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A) were markedly increased when they were carried out at pH 6.2 instead of pH 7.4. Cytotoxicity induced by phorbol myristate acetate (PMA), on the contrary, was significantly decreased at pH 6.2. It is noteworthy that cytotoxic responses induced by IC, zymosan and Con A were also increased when, 2 h after effector cell stimulation at pH 6.2, cytotoxicity was measured at pH 7.4. Finally, when we examined possible mechanisms involved in the augmentation of cytotoxicity, we observed that the oxidative response of IC-stimulated neutrophils, measured as chemiluminescence emission, was not increased at pH 6.2, on the contrary, it was significantly decreased. The relevance of these results is discussed.     

Enhancement of growth ofMycobacterium malmoense by acidic pH and pyruvate

The growth ofMycobacterium malmoense is dysgonic and slow on ordinary mycobacterium media. The effect of pH and pyruvate on the growth of ten strains was studied on a modification of Löwenstein-Jensen medium. Growth appeared sooner and was more abundant at pH<6.5. At pH 7 or higher, it was scarcely or not at all visible after six weeks of incubation. Pyruvate enhanced the growth of five strains that grew only poorly on glycerol-containing medium, even at acidic pH. The parallel use of both pyruvate and glycerol-containing media, pH 6 to 6.5, and an incubation period of seven weeks or longer are recommended for the isolation ofMycobacterium malmoense on Löwenstein-Jensen medium.     

Some low molecular weight proteins of bacteriophage lambda are produced by proteolytic degradation.

The effect of partial acid hydrolysis, boiling at neutral pH, and treatment with proteases on the sodium dodecyl sulfate (SDS)-polyacrylamide pattern of protein bands from phage λ and λ-related structures has been investigated. It was found that all of these treatments cleave the major proteins (particularly pE) in a reproducible way to produce smaller polypeptides. These polypeptides are also produced by the treatment normally used to prepare phage proteins for SDS-polyacrylamide gel electrophoresis.     

Effects of pH on the comparative culturability of fungi and bacteria from acidic and less acidic forest soils

Under aerobic conditions, the culturable microbial population of acidic forest soils was more tolerant to acidic cultivation conditions than was the culturable microbial population of less acidic soils. The number of culturable bacteria decreased sharply under acidic cultivation conditions, while the number of culturable fungi remained relatively constant over the pH range 2.2–6.5. The ratios of culturable bacteria to culturable fungi were greater than one at pH 6.5; in contrast, the bacteria-to-fungi ratios were less than one at pH 2.2–4. At pH's approximating those of the soils examined, culturable fungi predominated the culturable microbial community in acidic soils. However, relative to the populations resolved, acidic forest soils displayed a more acid tolerant bacterial population than did less acidic forest soils. The culturable fungal population contained both filamentous and yeast morphologies. An acid-tolerant fungal isolate that grew at pH 1 was identified as a subspecies of Penicillium frequentans, and an acid-tolerant yeast that grew at pH 2 was identified as the yeast stage of the basidiomycetes Ustilago maydis.     

Effects of extracellular pH upon the insulinotropic action of alpha-D-glucose pentaacetate.

The pentaacetate ester of alpha-D-glucose was recently introduced as a new insulin secretagogue. Its insulinotropic action appears mainly attributable to the catabolism of its hexose moiety in islet cells, but a direct effect of the ester itself upon a receptor apparently displaying analogy with that involved in the recognition of bitter agents by taste buds may also be operative. In the present study, the secretory response of rat isolated pancreatic islets to alpha-D-glucose pentaacetate (1.7 mM) was found to be preserved, except in the absence of any other exogenous nutrient, when the extracellular pH was raised from about 7.4 to 8.0. Inversely, however, when the extracellular pH was lowered to about 7.0, alpha-D-glucose pentaacetate inhibited both basal and D-glucose- or L-leucine-stimulated insulin output. These findings are interpreted to support a dual mode of action of alpha-D-glucose pentaacetate upon insulin secretion, a lowering of extracellular pH revealing a negative component of the islet B-cells functional response to such a monosaccharide ester.     

MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells

We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock‐out (MID1^?/?) mice have a 25–30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild‐type (WT) mice. We wondered why the MID1 gene knock‐out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1^?/? CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1^?/? CTL upregulated MID2 two–twenty‐fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1^?/? CTL completely rescued exocytosis of lytic granules in MID1^?/? CTL, and vice versa, knock‐down of MID2 inhibited exocytosis of lytic granules in both WT and MID1^?/? CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL.     

Phagolysosomes of Coxiella burnetii-infected cell lines maintain an acidic pH during persistent infection.

Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that multiples within vacuoles of phagolysosomal origin. Persistently infected cell lines were maintained in continuous culture for months. We studied the pH of the phagolysosomes by using two murine cell lines during early propagation of the bacteria and after establishment of persistent infection. Three strains of C. burnetii were studied because of the purported propensity of each strain to cause acute or chronic disease and to be resistant or susceptible to antibiotics. The pHs were calculated from fluorescence experiments with fluoresceinated dextran as a lysosomal probe. Phagolysosomal vacuoles maintained an acidic pH during a 36-day infection. Minimal variation of the pH occurred over the duration of the experiment with strains that caused either acute or chronic disease. Phagolysosomal pH remained stable for as long as 153 days with the Nine Mile phase II isolate. Thus, neither the course of C. burnetti infection nor the diversity of antibiotic susceptibility of the strains is related to variations in the phagolysosomal pH.     

Alpha 1-antitrypsin is present within the primary granules of human polymorphonuclear leukocytes.

Elastase is a potent proteolytic enzyme found within human neutrophil primary granules. Its major inhibitor in the serum is alpha 1-antitrypsin, a protein that is synthesized by hepatocytes but which has recently also been shown to be synthesized by circulating neutrophils. The authors have therefore carried out an immunocytochemical study at the light microscopic and ultrastructural level to determine the intracellular localization of alpha 1-antitrypsin. Double labeling with colloidal gold showed that alpha 1-antitrypsin is localized at the same site as neutrophil elastase, i.e., within primary granules. Secondary granules (detected by labeling for lactoferrin) were unstained for alpha 1-antitrypsin. Elastase and its major inhibitor therefore coexist within the same granule population within human neutrophils. Some difference in their intraorganelle distribution existed at the ultrastructural level (in that elastase tended to be localized at the periphery of the granules whereas alpha 1-antitrypsin was usually diffusely present in the matrix of the granules), but further studies are required to determine whether the two molecules are already complexed with each other within the neutrophil.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.     

Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH.

A keratinolytic proteinase with enzyme activity at acidic pH was isolated from culture filtrates of Trichophyton mentagrophytes, a major pathogenic fungus of dermatophytosis. The molecular weight of the proteinase was estimated to be 41,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 38,000 by gel filtration. The isoelectric point was determined to be 3.9. The proteinase had a pH optimum of 4.5 for keratin and 5.5 for hemoglobin. This enzyme hydrolyzed the synthetic chymotrypsin substrate Suc-Ala-Ala-Pro-Phe-MCA (Km, 0.59 mM), and its activity was strongly inhibited by chymostatin. Previously reported proteinases from dermatophytes have had enzyme activities in neutral or alkaline pH; however, healthy skin has a weakly acidic pH. Thus, the purified proteinase which has an optimal activity at acidic pH and hydrolyzes skin constituents could be an important virulence factor in dermatophytosis.